In the field of microbial production of compounds of interest, there is in general a growing desire to use recombinant microorganisms containing as little as possible of foreign DNA. Ideally the transformed microorganism would contain only the desired modifications at the gene(s)-of-interest and as little as possible or remnants of other DNA fragments used during the transformation experiment or during cloning.
Patent applications EP 635,574 and WO 9706261 reveal genes encoding phenotypically selectable markers, which can be removed once the gene(s)-of-interest are stably maintained in the organism. The examples in these patent applications are genes encoding acetamidases, which upon expression in a transformed cell enable the cell to grow on media with acetamide as the sole carbon and/or nitrogen source. This selectable marker has several advantages: it is a dominant but non-antibiotic marker, and it can be used even in fungi with an endogenous amdS gene, like Aspergillus nidulans (Tilburn et al., 1983, Gene 26: 205-221). Moreover, this selectable marker is a so-called bi-directional marker, meaning that besides the positive selection for its presence (forward selection) also a reverse selection selecting for the absence of the gene can be applied, using fluoroacetamide (see FIG. 1). This is successfully applied in species from the genera Aspergillus, Penicillium, Saccharomyces and Trichoderma. 
The amdS gene of Aspergillus nidulans is successfully used as a selection marker for different fungal species, for instance in Aspergillus niger and Penicillium chrysogenum. In addition, homologous amdS genes were described, obtained from Aspergillus niger for transformation of Aspergillus niger and obtained from Penicillium chrysogenum for transformation of Penicillium chrysogenum (WO 9706261). However, these amdS genes still have a major drawback. The forward selection, e.g. selection for the presence of the selectable marker gene, works, although for one of the genes the present invention will show that this is not the case. The problems become apparent when the reverse selection is applied, e.g. selection for the absence of the selectable marker gene. The most widely used (acet)amidase expression cassette, PgpdA-amdS from Aspergillus nidulans can give rise to isolation of false negatives, e.g. isolates which suggest by phenotype that they are devoid of the selection marker as a result of the negative selection protocol, but actually have the selection marker gene or fragments thereof stably maintained in the genome. For some reason they escape the selection pressure. This can be a burden in strain improvement programs where repeated transformations have to be performed, especially in those cases where a high throughput is required. Therefore, a bi-directional selection marker gene that functions 100% correctly in both directions, e.g. the forward and the reverse selection, is not available but is highly desirable.
The present invention surprisingly shows that the reverse selection, i.e. the deletion of an amidase marker gene from microbial strains, works with 100% efficiency when using the amidase encoding genes according to the invention. As a result, sequential modification of industrial production strains is feasible with high efficiency and throughput.